Toxoplasma gondii is an opportunistic parasite that infects one-third of the global population and is among the most frequent causes of central nervous system (CNS) infection complicating AIDS. T. gondii establishes a chronic infection in the CNS, and immune compromise due to HIV infection leads to parasite reactivation and fatal encephalitis. This proposal addresses a poorly understood aspect of parasite pathogenesis during clinical AIDS - how does T. gondii migrate through the brain? Evidence suggests that T. gondii infection of migratory leukocytes facilitates the colonization of vital organs such as the brain via a 'Trojan horse' mechanism. Monocytes in particular are highly motile cells and a target for T. gondii infection. Our strong preliminary data demonstrate that T. gondii infection of human monocytes induces a hypermigratory phenotype and dysregulates the function of key adhesion molecules called integrins. The objective of this proposal is to define how T. gondii induces monocyte motility and disseminates through the brain. The central hypothesis is that T. gondii spreads through tissues by co-opting the migratory potential of infected cells. Two aims are proposed to test the hypothesis: 1) Determine how T. gondii enhances the migration of infected cells, and 2) Define how T. gondii disseminates through the infected brain. In the first aim, we will investigate the regulation of integrin signaling and focal adhesion formation in infected primary human monocytes from healthy donors and from HIV patients. We will determine the role of focal adhesion kinase (FAK) by using monocytes expressing mutant variants of FAK that are constitutively active or non-phosphorylatable (inactive). We will also determine the interactions of the scaffolding molecules talin and paxillin with FAK and the dynamics of their assembly and disassembly in infected monocytes during migration. In the second aim, we will use intravital two-photon imaging and an animal model for T. gondii infection to define the myeloid cell type(s) that are infected and potentiate parasite spread through the mouse brain. Transgenic mice that express fluorescently labeled monocytes or microglia will be infected with T. gondii. The dynamics of their migration through the parenchyma will be determined during acute infection and in chronically infected mice undergoing parasite reactivation due to immune suppression. All of the necessary reagents are on hand in the lab or commercially available, and all the techniques, including biochemical and migration assays, total internal reflection fluorescence (TIRF) microscopy, and intravital imaging, are currently employed by our research team. The significance of this research is that understanding T. gondii dissemination may allow for therapeutic targeting of this process to mitigate disease during AIDS. The proposal is innovative because it applies a multidisciplinary approach and novel tools to identify the cellular and molecular mechanisms that mediate T. gondii dissemination in vitro and in vivo, which will ultimately shed light on a key aspect of parasite pathogenesis in HIV/AIDS patients.